Abstract In the brain, the transcription factors MeCP2 and CREB play crucial roles in activity-dependent gene regulation for long-term memory formation and learning. Dysfunction of these proteins is associated with brain disorders such as Rett syndrome and drug addiction. MeCP2 was originally identified as a transcriptional repressor that specifically recognizes methylated CpG dinucleotides in DNA. More recently, despite the lack of any known transcriptional activation domain, it was found that neuronal MeCP2 activates more than 2,000 genes under the control of CpG island promoters where DNA methylation is rare. The overall goal of this R21 project is to elucidate the mechanisms by which MeCP2 acts as a transcriptional activator. The central hypothesis tested in this project is that MeCP2 activates many genes by novel dual mechanisms through the direct interaction with CREB and through indirect interplay that prevents CREB1 from being sequestered in nonfunctional decoys. This research project will pursue the following specific aims: 1) characterize the direct interaction between CREB and MeCP2 in vitro and 2) unravel the indirect interplay between these proteins via DNA methylation. The research team will use biophysical and biochemical approaches to examine the dual mechanisms. New knowledge about transactivation by MeCP2 will provide insight into epigenetic regulation in the brain and deepen our understanding of brain disorders. For example, our biophysical characterizations of the CREB-MeCP2 interaction will provide new perspectives for interpreting Rett syndrome mutations of MeCP2. The approaches established through this project would potentially facilitate pharmacological screening of compounds that inhibit the CREB-MeCP2 interaction for drug addiction therapeutics in the future.